Many diseases, including several forms of cancer, depend on the inappropriate expression of pathological proteins, such as oncogenes. Frequently, the structure-function of these proteins belies a modular construction, with an essential functional domain in the C-terminal portion of the protein (for example the transactivation domain of a transcription factor). Mutants lacking the C-terminal domain can have altered and antagonistic functions, and in some cases they can behave as dominant- negative variants. Such variants are sometimes naturally generated by alternative splicing/polyadenylation. However, these are not always present or not easily activatable. A method to specifically induce these dominant negative variants would be highly desirable and have a significant therapeutic potential in a broad range of diseases, including cancer. In order to generate a pre-designed, desirable truncated variant instead of the pathological one we propose development of a novel universal methodology, based on a brand new design of antisense molecules that are able to redirect termination of translation from the normally translated full-length ORF to an upstream specified location. These Self-Wrapping Antisense Translation Termination (SWATT) compounds work by introducing an insurmountable steric block that cannot be resolved by the helicase activity of incoming translating ribosomes, which can normally unwind extensive secondary structures within the pre-mRNA. This roadblock would thus result in ribosome drop-off and expression of a shorter, C-terminally truncated variant. We will validate the viability of this novel strategy by assessing SWATT compounds on specifically designed reporter luciferase constructs and, eventually, by showing their biological relevance using Notch signaling as a proof-of-principle substrate in cancer cells. Induction of a C-Terminal dominant negative version of Notch, lacking the intracellular signaling domain, should inhibit Notch signaling by engaging Notch ligands in non-productive interactions, and thus prevent its oncogenic functions. SWATT compounds will provide an additional tool to dissect the structure-function properties of proteins in their physiological contexts, and they wil constitute the basis for a novel therapeutic approach in the treatment of cancer and other diseases. PUBLIC HEALTH RELEVANCE: We plan to develop a novel technology to re-direct the production of proteins that contribute to tumor progression to variants of the same proteins with opposite, anti- tumorigenic properties (Dominant-Negative variants). This will be obtained by introducing a physical 'road-block' into the path of ribosomes during translation at specific, pre-determined points, and therefore forcing them to interrupt protein synthesis there. If successful, this work might constitute the basis for a novel, alternative approach to cancer therapy, with potential application to a broad range of tumors.